RESEARCH ARTICLE 3813
Development 138, 3813-3822 (2011) doi:10.1242/dev.064816
© 2011. Published by The Company of Biologists Ltd
Integrins are necessary for the development and
maintenance of the glial layers in the Drosophila peripheral
nerve
Xiaojun Xie and Vanessa J. Auld*
SUMMARY
Peripheral nerve development involves multiple classes of glia that cooperate to form overlapping glial layers paired with the
deposition of a surrounding extracellular matrix (ECM). The formation of this tubular structure protects the ensheathed axons
from physical and pathogenic damage and from changes in the ionic environment. Integrins, a major family of ECM receptors,
play a number of roles in the development of myelinating Schwann cells, one class of glia ensheathing the peripheral nerves of
vertebrates. However, the identity and the role of the integrin complexes utilized by the other classes of peripheral nerve glia
have not been determined in any animal. Here, we show that, in the peripheral nerves of Drosophila melanogaster, two integrin
complexes (PS2PS and PS3PS) are expressed in the different glial layers and form adhesion complexes with integrin-linked
kinase and Talin. Knockdown of the common beta subunit (PS) using inducible RNAi in all glial cells results in lethality and glial
defects. Analysis of integrin complex function in specific glial layers showed that loss of PS in the outermost layer (the
perineurial glia) results in a failure to wrap the nerve, a phenotype similar to that of Matrix metalloproteinase 2-mediated
degradation of the ECM. Knockdown of PS integrin in the innermost wrapping glia causes a loss of glial processes around axons.
Together, our data suggest that integrins are employed in different glial layers to mediate the development and maintenance of
the protective glial sheath in Drosophila peripheral nerves.
INTRODUCTION
The development of the glial sheath in peripheral nerves is vital to
provide structural support, insulate and protect axons from physical
damage and pathogens. The architecture of peripheral nerves
consists of multiple glial layers and a surrounding basal lamina.
Centermost in vertebrate nerves are the myelinating or nonmyelinating Schwann cells that wrap individual axons or bundles
of axons, respectively. Groups of myelinated or non-myelinated
nerve fibers are initially encased by a basal lamina, later becoming
embedded in a collagenous connective tissue, called the
endoneurium, to form fascicles. Each fascicle is sheathed by the
perineurium, a protective barrier of overlapping squamous-like
perineurial cells (Olsson, 1990). Similarly, the Drosophila larval
peripheral nerve is made up of several distinct layers (Stork et al.,
2008). The innermost wrapping glia (WG) separate and ensheath
axons in a manner similar to vertebrate non-myelinating Schwann
cells. The WG are next surrounded by subperineurial glia (SPG),
which form intercellular septate junctions and create the bloodnerve barrier (Stork et al., 2008). The outermost cell layer is a
monolayer of squamous-like perineurial glia (PG) (Lavery et al.,
2007). The final layer is the neural lamella (NL), a dense basal
lamina encasing each peripheral nerve. Drosophila and vertebrate
peripheral glia express many of the same proteins, such as NCAM
and L1 (Freeman and Doherty, 2006) and homologs of the
paranodal junction proteins (Bhat, 2003), and use many of the same
Department of Zoology, Cell and Developmental Biology, University of British
Columbia, Vancouver, BC, V6T 1Z3, Canada.
*Author for correspondence ([email protected])
Accepted 27 June 2011
developmental programs, such as Erb/neuregulin signaling
(reviewed by Parker and Auld, 2006; Newbern and Birchmeier,
2010).
In vertebrates, the basal lamina contains extracellular matrix
(ECM) components known to be important for glial development.
For example, laminins, a major ECM component, are essential for
Schwann cell differentiation, axon sorting and myelination (Chen
and Strickland, 2003; Wallquist et al., 2005; Yang et al., 2005; Yu
et al., 2005). Similarly, non-myelinating Schwann cells lacking
laminins fail to differentiate and the associated C-fibers are lost (Yu
et al., 2009). These ECM signals are transduced by specific
receptors, which include Dystroglycan, Glypican and integrins
(Feltri and Wrabetz, 2005). Integrins are the best-characterized
ECM receptors in Schwann cells and consist of one alpha and one
beta subunit. Loss of 1 integrin or integrin-linked kinase (Ilk) in
myelinating Schwann cells results in defects in radial sorting and
myelination (Feltri et al., 2002; Fernandez-Valle et al., 1994;
Pereira et al., 2009). Although there is evidence for a role of
integrin adhesion and signaling in myelinating Schwann cells, the
role of integrins in the development of the non-myelinating
Schwann cells or of the perineurium is not understood.
Complicating the investigation of integrins in peripheral nerve
development is the complexity of integrin heterodimer expression
in vertebrates (Feltri and Wrabetz, 2005). By contrast, Drosophila
melanogaster has a relatively simple family of integrin subunits
consisting of five alpha and two beta subunits (Brown et al., 2000).
Therefore, we used Drosophila to investigate the role of integrins
and ECM interactions during the development of the glial layers of
the peripheral nerve. Here, we show that specific integrin
heterodimers play a role in the development and maintenance of
the glial layers. Downregulation of integrin expression results in
wrapping defects in both the PG and WG. Furthermore, the basal
DEVELOPMENT
KEY WORDS: Glia, Integrins, Peripheral nervous system development, Drosophila
3814 RESEARCH ARTICLE
Development 138 (17)
lamina is essential for the proper maintenance of the perineurial
wrap. Collectively, our results demonstrate that Drosophila
integrins and the basal lamina are essential for proper glial sheath
development and maintenance in the peripheral nerve.
MATERIALS AND METHODS
Fly strains and genetics
Immunohistochemistry and imaging analysis
The following primary antibodies were obtained from the Developmental
Studies Hybridoma bank (NICHD, University of Iowa): mouse anti-PS
(CF.6G11) (Brower et al., 1984) used at 1:10; mouse anti-PS2 (CF.2C7)
(Brower et al., 1984) at 1:5; and mouse anti-Repo (8D12) (Alfonso and
Jones, 2002) at 1:50. Other primary antibodies were: rabbit anti-PS3
(Wada et al., 2007) used at 1:200; rabbit anti-HRP (horseradish peroxidase;
Jackson ImmunoResearch) at 1:500; and rabbit anti-Drosophila -laminin
(LanB2) (Abcam) at 1:100. All secondary antibodies were used at 1:200:
goat anti-mouse Alexa 568 and Alexa 647; goat anti-rabbit Alexa 568 and
Alexa 647 (Molecular Probes/Invitrogen).
Dissection and fixation for immunofluorescence were performed
according to standard procedures (Sepp et al., 2000). Unless specified
otherwise, images were obtained with a DeltaVision Spectris (Applied
Precision) using a 60⫻ objective (NA 1.4) with 0.2 m z-sections. Image
stacks were deconvolved and rotated with SoftWorx (Applied Precision)
based on measured point spread functions of 0.2 m fluorescent beads
(Molecular Probes) mounted in Vectashield (Vector Laboratories). Images
were exported to Photoshop and Illustrator CS4 (Adobe) for compilation.
For lower magnification images, images were captured using a 20⫻
objective (NA 0.4, DeltaVision) or a 10⫻ objective (NA 0.3, Axioskop 2,
Zeiss).
PG numbers were counted in nerves from abdominal segment 8 (A8)
from late third instar larvae. The mean and the standard deviation were
calculated in Excel 2010 (Microsoft).
Fig. 1. Axons are surrounded by three glial layers in the
Drosophila larval peripheral nerve. (A)Transverse section of a third
instar larval peripheral nerve. From external to internal are the neural
lamella (NL), perineurial glia (PG), subperineurial glia (SPG), wrapping
glia (WG) and axons (AX). Two PG nuclei are shown (green).
(B-F)Orthogonal sections from third instar nerves illustrate the GFPtagged proteins and GAL4 drivers used to label the ECM and the
different cellular glial layers. Panels have been digitally expanded. The
NL was labeled using Perlecan-GFP (B, green). PG were labeled using
46F-GAL4::CD8-RFP (B and C, red) and Jupiter-GFP (C and D, green).
SPG were labeled using Gli-GAL4::CD8-RFP (D and F, red). WG were
labeled using Nrv2-GAL4::CD8-GFP (E, green) and Nrv2-GFP (F, green).
Axons were immunolabeled using an anti-HRP antibody (E, red; F, blue).
Glial nuclei are shown by DAPI labeling (blue), except in C where the
SPG nucleus is immunolabeled with a Repo antibody (blue). Scale bars:
5m.
1). In the center of the peripheral nerve are the WG that ensheath
individual axons and axonal bundles. WG express Nervana 2
(Nrv2)-GAL4 (Fig. 1E) and the Nrv2 protein tagged with GFP
(Nrv2-GFP) (Fig. 1F, Table 1).
Table 1. Summary of markers for ECM and glial layers in the
peripheral nerve
Glia subtypes
NL
RESULTS
In the larval peripheral nerve, axons are wrapped by three
consecutive layers of glia: the outermost perineurial glia (PG),
intermediate subperineurial glia (SPG) and inner wrapping glia
(WG) (Fig. 1A) (Stork et al., 2008). The dense basal lamina (the
neural lamella, NL) that surrounds the nerve can be visualized with
the proteoglycan Perlecan endogenously tagged with GFP (Fig.
1B). The PG are located just below the NL and specifically express
the enhancer-trap line 46F-GAL4 as well as Jupiter-GFP, a
microtubule-associated protein (Karpova et al., 2006)
endogenously tagged with GFP (Fig. 1C,D) (Table 1). Directly
below the perineurial layer are the SPG, which express either SPGGAL4 (Stork et al., 2008) or Gliotactin (Gli)-GAL4 (Fig. 1D, Table
PG
SPG
WG
X
X
X
X
GAL4 drivers
repo-GAL4
46F-GAL4
SPG-GAL4
Gli-GAL4
Nrv2-GAL4
X
X
X
GFP trap lines
perlecan-GFP
viking-GFP
Jupiter-GFP
nrv2-GFP
X
X
X
X
NL, neural lamella; PG, perineurial glia; SPG, subperineurial glia; WG, wrapping glia;
Gli, Gliotactin; Nrv2, Nervana 2.
DEVELOPMENT
The following fly strains were used: repo-GAL4 (Sepp et al., 2001); SPGGAL4 (Schwabe et al., 2005); nrv2-GAL4 (Sun et al., 1999); Gli-GAL4
(Sepp and Auld, 1999); 46F-GAL4 (a gift from Dr Yong Rao, McGill
University); UAS-mCD8-GFP (Lee and Luo, 1999); UAS-mCD8-RFP (a
gift from Dr Elizabeth Gavis, Princeton University); UAS-Mmp2 (PageMcCaw et al., 2003); UAS-Dicer2 (Dietzl et al., 2007); tubP-GAL80ts
(McGuire et al., 2003); mys1 (Bunch et al., 1992); FRT19A,tubPGal80,hsFLP,w* (Lee and Luo, 1999); repo-FLP (Stork et al., 2008); and
UAS-nls-GFP (a gift from Dr Douglas Allan, University of British
Columbia). The following GFP protein-trap insertions were used: perlecanGFP; Jupiter-GFP; nrv2-GFP; Ilk-GFP; talin-GFP (Kelso et al., 2004;
Morin et al., 2001). UAS-RNAi strains were obtained from the VDRC
(Austria) (Dietzl et al., 2007), NIG (Japan) and TRiP (Harvard). RNAi
experiments were carried out at 25°C and with UAS-Dicer2 in both control
and experimental crosses unless specified. Control and PS MARCM
clones were generated using female FRT19A; +/+; repo-GAL4, UAS-CD8GFP/TM6B, Tb or mys1, FRT19A/FM7; +/+; repo-GAL4, UAS-CD8GFP/TM6B, Tb with male FRT19A, tubP-Gal80, hsFLP, w*/Y; +/+; repoFLP.
Fig. 2. PS integrin is expressed in the peripheral glia layers.
Immunolabeling for the PS integrin subunit in Drosophila third instar
nerves expressing either Ilk-GFP or different glial markers in single
0.2m z-sections. (A)PS (red), Ilk-GFP (green) and DAPI (blue)
labeling. Both PS and Ilk form puncta and are associated with each
other in the peripheral nerve. (B-G)PS labeling in the different glial
subtypes: PG using Jupiter-GFP (B-D, green), SPG using Gli-GAL4::CD8RFP (B-G, red) and WG using Nrv2-GFP (E-G, green). The labeled glial
layers are also illustrated to the right. The red boxes in B and E were
digitally expanded as shown in C and F, respectively. The green lines in
B and E indicate the positions of the orthogonal sections in D and G,
respectively. PS integrin was found in the PG outer membrane (C and
D, arrow), the PG-SPG boundary (C and D, arrowhead), the SPG inner
membrane (F and G, arrowhead) and the WG membrane (F and G,
arrows). Scale bars: 10m in A,B,E; 5m in C,D,F,G.
Specific integrins are located in the glial cells of
the larval peripheral nerve
We began our investigation of the role of integrins in peripheral
nerve development by examining which integrins are expressed
by the glia. We were unable to detect integrin expression in
embryonic glia by genetic or immunofluorescence analysis (data
not shown). Thus, our analysis concentrated on larval peripheral
glia and on those integrin subunits with known vertebrate
homologs (PS, PS1, PS2, PS3) (Brown et al., 2000). All
three classes (PG, SPG and WG) expressed integrin complexes
when assayed with an antibody to the beta subunit, PS (Mys)
(Fig. 2). In addition, we observed immunolabeling with
antibodies to two alpha subunits, PS2 and PS3 (Fig. 3), but
not PS1 (data not shown). The integrin subunits were observed
in discrete puncta or stripes and were associated with Ilk-GFP
(Fig. 2A, Fig. 3A, see Fig. S1 in the supplementary material) and
RESEARCH ARTICLE 3815
Fig. 3. PS2 and PS3 integrins are expressed in different glial
layers of the larval peripheral nerve. Drosophila third instar larval
nerves immunolabeled with antibodies to PS2 (A, red; B-D, blue) and
PS3 (A,E-G, blue) integrins in single 0.2m z-sections. (A)Ilk-GFP
(green) and integrin alpha subunit PS2 (red) and PS3 (blue)
immunolabeling. Both alpha subunits form puncta and are associated
with Ilk in the peripheral nerve. (B-G)Specific glial layers were labeled
with different markers: PG using Jupiter-GFP (B-D, green), SPG using
Gli-GAL4::CD8-RFP (B-G, red) and WG using Nrv2-GFP (E-G, green).
The labeled glial layers are also illustrated to the right. The red boxes in
B and E were digitally magnified as shown in C and F, respectively. D
and G are orthogonal sections of z-stacks at the positions indicated by
green lines in B and E, respectively. (B-D)PS2 integrin labeling is mostly
found in the outer glial cells, the PG outer membrane (arrows), the PGSPG boundary (arrowheads) and in the SPG inner membrane (asterisks).
(E-G)PS3 integrin labeling is mostly found in the internal glial layers, in
the SPG inner membrane (arrowheads) and in the WG membrane
(arrows). PS3 integrin labeling was often associated with the SPG
membrane protrusions (asterisks). Scale bars: 10m in A,B,E; 5m in
C,D,F,G.
Talin-GFP (see Fig. S2A-C in the supplementary material). The
GFP fusion proteins only partially overlapped with PS integrin;
compare the puncta (arrowheads) between the two focal planes
Z31 (see Fig. S1B in the supplementary material) and Z34
(see Fig. S1C in the supplementary material), which do overlap
in an orthogonal section of the nerve (arrows in Fig. S1D in the
supplementary material). This was not unexpected as Ilk and
Talin form adhesion complexes with integrins through binding
to the intracellular domain of beta integrin (Delon and Brown,
2007) and the integrin antibodies are thought to bind the
extracellular domain of these transmembrane receptors (Brower
DEVELOPMENT
Integrins and glial development
et al., 1984). The colocalization of the integrin subunits with Ilk
and Talin in the larval nerve suggests that adhesion complexes
are found throughout the glial layers of the peripheral nerve.
The beta integrin subunit is expressed in all glial
cell layers
To address which glial layers express different integrin complexes,
we used a combination of proteins endogenously tagged with GFP
and GAL4 drivers to label the individual glial layers (Fig. 1; Table
1). First, we examined the distribution of the common PS subunit,
which is able to form heterodimers with all alpha subunits. PG
were labeled with Jupiter endogenously tagged with GFP in
conjunction with SPG labeled using Gli-GAL4 driving the
expression of UAS-CD8-RFP (Gli::CD8-RFP). Immunolabeling of
PS integrin was observed on the PG outer membrane (Fig. 2C,D,
arrows) suggesting an interaction of the integrin complex with the
external ECM/NL (Stork et al., 2008). PS integrin
immunolabeling was also found between Jupiter-GFP and
Gli::CD8-RFP (Fig. 2C,D, arrowheads), suggesting that these
integrin proteins also localize to the PG and SPG boundary.
The PS was also observed in the nerve, internal to the CD8RFP-labeled SPG (Fig. 2C,D), in the areas occupied by WG and
axons. To study whether the internal PS integrin is located at the
WG membrane, Nervana 2 endogenously tagged with GFP (Nrv2GFP) (Morin et al., 2001) was used to label the WG (Fig. 2E-G).
At the same time, Gli::CD8-RFP was used to label the SPG
membrane. PS immunolabeling was consistently found to
associate with both the Gli::CD8-RFP-labeled SPG (Fig. 2F,G,
arrowheads) and with Nrv2-GFP. PS was often observed between
Nrv2-GFP and protrusions of CD8-RFP (Fig. 2F,G, arrows) and
thus might mediate interactions between the SPG and WG.
Importantly, all of the PS integrin observed in the peripheral
nerve appears to be expressed by glial cells and not neurons. We
confirmed this by knockdown of PS expression using PS-RNAi
specifically in the glia cells (see below). Our data indicate that PS
integrin is expressed by the glial cells and forms adhesion
complexes in the glial membranes in the larval peripheral nerve.
Differential glial expression of integrin alpha
subunits
Both the PS2 and PS3 integrin subunits were located to
adhesion complexes with Ilk-GFP and Talin-GFP in the larval
peripheral nerve and were expressed with different intensities in the
different glial layers (see Fig. S1E-G in the supplementary
material). The specific glial layer markers outlined above were
used to further characterize the discrete localization of the alpha
subunits (Fig. 3). PS2 integrin immunolabeling was found on the
external surface of the nerve, outside of the Jupiter-GFP-labeled
PG, similar to PS (Fig. 3B-D, arrows). This further supports a role
for the integrin complex in mediating adhesion between the PG and
the ECM. PS2 was also found between Jupiter-GFP and
Gli::CD8-RFP (Fig. 3C,D, arrowheads), indicating that PS2 is
also localized at the PG and SPG boundary. A few PS2-positive
puncta were also found in the center of the nerve, internal to
Gli::CD8-RFP (Fig. 3C,D, asterisks), suggesting that PS2 might
be expressed at low levels by the internal WG or by extensions of
the SPG into the center of the nerve.
Conversely, most of the PS3 integrin immunolabeling was
found in the center of the nerve internal to the Gli::CD8-RFP (Fig.
3F,G, arrows and arrowheads). PS3 was located between the
RFP-labeled SPG processes and Nrv2-GFP-labeled processes.
Moreover, PS3 was associated with CD8-RFP-labeled
Development 138 (17)
membranes seen protruding between the internal Nrv2-GFPlabeled members of the PG (Fig. 3F,G, asterisks), suggesting that
PS3 might be involved in an interaction between SPG and WG.
However, we cannot rule out the possibility that PS3 expression
might indicate an interaction between the WG and their associated
axons. In general, our data suggest that the PS2 and PS3 integrin
subunits are both expressed by the different glial layers and might
be important for glia-ECM and glia-glia interactions.
Integrin function is necessary in larval peripheral
glia
The question then arises, what functions do integrins have in the
peripheral glia? Null mutants of PS (mys) are embryonic lethal and
we were unable to detect PS immunolabeling in embryonic glia
(data not shown). In mys mutant embryos, we were unable to detect
any defects in glia cell migration (data not shown). This, paired with
a lack of peripheral axon defasciculation, which is a hallmark of
disruption in glial ensheathment, suggests that integrin function
occurs at later developmental stages. Therefore, we took two
approaches to remove PS function from peripheral glia during
larval development: MARCM analysis and RNA interference
(RNAi). To generate mosaic clones for a mys null in peripheral glia
we used the MARCM technique and assayed glial development in
third instar larvae. CD8-GFP-labeled mys clones were seen in the
peripheral (Fig. 4A, arrows) and central (Fig. 4A, arrowhead)
nervous systems. Using this approach, the majority of glial clones
were in the PG (14/14 in control larvae and 14/16 in mutant larvae).
In control wild-type clones, the CD8-GFP-labeled PG formed a thin
layer on the outside of the HRP-labeled axons (Fig. 4B), resulting in
an intact circle around the nerve (Fig. 4D). In controls, PS
immunolabeling was consistently found in the perineurial membrane
(Fig. 4C,D, arrows) and internal glial layers. In the mys null clones,
PS labeling was absent from the GFP-labeled perineurial membrane
(Fig. 4F,G, arrowheads) but still present in the internal wild-type
SPG and WG layers. Loss of PS integrin resulted in PG that failed
to form a uniform sheath, such that CD8-GFP-labeled membranes
were observed only on one side of the nerve (Fig. 4E) and failed to
encircle the nerve (Fig. 4G). It is important to note that the CD8GFP-labeled membranes of the PS mutant PG still covered a
significant distance along the length of some nerves suggesting that,
without PS integrin, PG were able to send out processes along the
nerve. Although MARCM analysis revealed the importance of PS
integrin in larval PG development, this approach had two
disadvantages. First, the majority of glial clones were in the PG. SPG
(n0) and WG (n2) clones were rare, as these glial subtypes do not
actively divide at later stages. Second, in each nerve with PG clones,
only a small population of PG were PS mutants, making it difficult
to evaluate the overall impact.
To overcome these limitations, we used RNAi (Tavernarakis et
al., 2000) to knock down integrins in all or individual glial
subtypes. RNAi lines known to target specific integrin subunits
(Perkins et al., 2010) were obtained; these target at least two
independent regions of each integrin subunit. We tested the ability
to knock down specific integrin subunits in glia using the repoGAL4 driver. Repo is a transcription factor that is exclusively
expressed in all glia except the midline glia (Xiong et al., 1994).
When repo-GAL4 was used to drive PS-RNAi, immunolabeling
by the PS antibody was decreased (Fig. 5B) as compared with
control nerves (Fig. 5A). The degree of PS integrin
immunolabeling in the underlying body wall muscles was used as
an internal control. This suggests that PS-RNAi is able to knock
down PS expression specifically in glia. In addition, the loss of
DEVELOPMENT
3816 RESEARCH ARTICLE
Fig. 4. MARCM analysis of a PS mutant in the larval peripheral
nerve. MARCM using flipase expression in glia was used to generate
wild-type or homozygous PS (mys) clones labeled with CD8-GFP.
(A)Control and mys glial clones were identified with CD8-GFP and
immunolabeled for Repo (red) and HRP (blue). At low magnification
(10⫻ objective), clones were seen in both peripheral nerves (arrows)
and the CNS (arrowheads). (B-G)High-magnification images of nerves
with control and mutant PG clones shown in single 0.2m z-sections
(B,E) and orthogonal sections of z-stacks (D,G). Green lines indicate the
positions of orthogonal sections. Boxed regions were digitally expanded
as shown in C and F. (B-D)In control clones, CD8-GFP labeling wraps
around the circumference of the nerve. Arrows indicate PS labeling
(red) in the PG outer membrane, as compared with the core of HRPlabeled axons (blue). (E-G)In mys clones, CD8-GFP labeling is found
only on one side of the HRP labeling in the z-section. The CD8-GFPlabeled membrane does not wrap around the nerve in the orthogonal
section. PS labeling is not found in the PG outer membrane
(arrowheads), but is still seen in the inner layers of glia within the nerve.
Scale bars: 10m in B,E; 5m in C,D,F,G.
PS labeling throughout the nerve in the repo::PS-RNAi larvae
suggests that PS integrin is expressed only by glial cells and not
by the neurons.
PS2-RNAi and PS3-RNAi lines were used to knock down
expression of the corresponding alpha subunits. In repo::PS2RNAi nerves, PS2 labeling was removed but the PS3 labeling
appeared normal (see Fig. S3B in the supplementary material).
Consistently, PS3 labeling, but not PS2 labeling, was greatly
reduced in repo::PS3-RNAi nerves (see Fig. S3C in the
supplementary material), indicating that the PS2-RNAi and
PS3-RNAi constructs were subunit specific. Labeling of muscles
and of trachea were used as internal staining controls for PS2 and
PS3, respectively.
RESEARCH ARTICLE 3817
Fig. 5. Expression of PS-RNAi in peripheral glia. (A,B)repo-GAL4
was used to express CD8-GFP (green) and PS-RNAi in all peripheral
glia. All panels are projections of the entire z-stack. PS labeling (red)
was greatly decreased in the RNAi nerve (B) when compared with
control nerve (A). As an internal control, PS labeling was also observed
in muscles and remained unchanged. CD8-GFP (green) was not evenly
distributed along the RNAi nerve (B) compared with the control nerve
(A). Axons were labeled with HRP (blue). (C-J)46F-GAL4 was used to
express CD8-GFP (green) and PS-RNAi in the PG. (C,G)Lower
magnification views (20⫻ objective) of control (C) and mutant (G)
nerves. Note the seamless coverage of GFP in control nerves (arrow)
and the discontinuity of GFP in mutant nerves (arrowhead). (D-F,H-J)
High-magnification images of control (D,F) and a mutant (H,J) nerves in
single 0.2m z-sections (D,H) and orthogonal sections of z-stacks (F,J).
Green lines indicate the positions of orthogonal sections. Boxed regions
were digitally expanded as shown in E and I. (D-F)In the control nerve,
CD8-GFP labeling wraps around the circumference of the nerve. Arrows
indicate PS labeling (red) in the PG outer membrane, as compared
with the core of HRP-labeled axons (blue). (H-J)In the 46F::PS-RNAi
nerve, CD8-GFP labeling is found only on one side of the nerve (HRPlabeled axons, blue) and fails to wrap the entire circumference.
Arrowheads point to PG outer membrane where no PS labeling is
found. The labeled glial layers and morphological changes are
illustrated to the right. N, nerve; M, muscle; VN, ventral nerve cord.
Scale bars: 50m in C,G; 10m in D,H; 5m in A,B,E,F,I,J.
DEVELOPMENT
Integrins and glial development
3818 RESEARCH ARTICLE
Development 138 (17)
Table 2. Severity of integrin and talin RNAi-induced phenotypes using 46F-GAL4
Category 0
No.
1
2
3
4
5
6
7
8
9
10
Drosophila line
w[1118] (control)
UAS-PS-RNAi (VDRC_GD)
UAS-PS-RNAi (NIG)
UAS-PS2-RNAi (VDRC_GD)
UAS-PS2-RNAi (NIG)
UAS-PS3-RNAi (VDRC_GD)
UAS-PS3-RNAi (NIG)
UAS-PS2-RNAi + UAS-PS3-RNAi
UAS-talin-RNAi (VDRC_GD)
UAS-talin-RNAi (NIG)
Category 1
Category 2
Total
n
%
n
%
n
%
69
64
59
63
53
61
57
39
51
65
67
0
2
53
49
59
54
3
0
0
97
0
3
84
92
97
95
8
0
0
2
2
12
10
4
2
3
9
16
3
3
3
20
16
8
3
5
23
31
5
0
62
45
0
0
0
0
27
35
62
0
97
76
0
0
0
0
69
69
95
Category 0, nerves in which no perineurial glia show wrapping defects; category 1, nerves in which a minority of perineurial glia show wrapping defects; category 2, nerves in
which the majority of perineurial glia show wrapping defects. VDRC_GD, GD constructs from Vienna Drosophila RNAi Center; NIG, fly stocks of the National Institute of
Genetics in Japan.
Integrin function is necessary for perineurial glia
wrapping
To study how loss of integrin affects the outer PG layer, 46FGAL4 was used to express the integrin RNAi constructs. In
control third instar larvae, 46F-GAL4-driven CD8-GFP formed a
seamless sheath around the exterior of the peripheral nerves (Fig.
5C,F). The coverage of 46F::CD8-GFP throughout the entire
surface of HRP labeling (see Movie 1 in the supplementary
material) suggests that the membranes from different perineurial
cells attach to each other and wrap around the nerve
collaboratively. In third instar larvae with 46F-GAL4 driving the
expression of PS-RNAi (46F::PS-RNAi), PS antibody
labeling was decreased in the PG (Fig. 5I,J, arrowheads) as
compared with controls (Fig. 5E,F, arrows). The PS labeling
appeared normal in the internal glia, indicating that PS integrin
expression was knocked down specifically in the PG.
PS RNAi caused similar morphological changes to the PG as
observed in the PS (mys) null MARCM clones. The CD8-GFPlabeled PG membrane was observed only on one side of the nerve
(Fig. 5H) and failed to encircle the nerve (Fig. 5J). Moreover,
individual PG cells became distinct (Fig. 5G) as the PG became
detached from each other (see Movie 2 in the supplementary
material).
Given that PS2 integrin labeling was mostly observed on both
sides of perineurial cells, we asked whether loss of PS2 integrin
would phenocopy the PS-RNAi. 46F-GAL4 was used to express
UAS-PS2-RNAi and similar PG wrapping phenotypes were
observed (data not shown). However, the penetrance of the
phenotypes was much lower in the 46F::PS2-RNAi larvae than in
the 46F::PS-RNAi larvae (Table 2). The severity of the defects
was also different. In 46F::PS2-RNAi larvae, all defective nerves
fell into category 1, in which a minority of PG failed to wrap,
whereas in 46F::PS-RNAi most of the defective nerves fell into
category 2, in which the majority of perineurial cells displayed a
wrapping failure. Given the efficiency of UAS-PS2-RNAi (see
Fig. S3B in the supplementary material), the difference between
46F::PS2-RNAi and 46F::PS-RNAi might be due to
compensation by another alpha subunit. As the PS3 integrin was
also found in the peripheral nerve, UAS-PS3-RNAi was coexpressed with UAS-PS2-RNAi using 46F-GAL4. As expected,
more nerves (92%) had PG that failed to wrap and most of them
(69%) were scored as category 2 (Table 2). This suggests that PS2
and PS3 integrins are functionally redundant in the PG, similar to
observations made concerning midgut formation (Martin-Bermudo
et al., 1999).
To determine whether PG wrapping requires the integrin
adhesion complex, we specifically knocked down Talin using
RNAi driven by 46F-GAL4 and observed wrapping defects similar
to those caused by integrin RNAi (Table 2, see Fig. S2D-F in the
supplementary material). Our data suggested that PG wrapping of
the peripheral nerve requires an integrin adhesion complex that
contains PS2PS or PS3PS integrin plus Talin.
To check whether the PG wrapping defect is due to an
insufficient number of PG to match the growing nerve surface, we
quantified the PG number in A8 nerves by co-expressing a nuclearlocalized GFP (UAS-nls-GFP) using 46F-GAL4. Control nerves
had 40±6 PG on average (n12). Knockdown of PS resulted in
either a reduction or an increase in PG nuclei depending on the
RNAi line used [13±3 (n11) for PS-RNAi (VDRC_GD) and
57±7 (n14) for PS-RNAi (NIG)]. However, knockdown of Talin
also showed an increase in the number of PG [50±7 (n12) for
Talin-RNAi (NIG) and 55±8 (n14) for Talin-RNAi
(VDRC_GD)]. As all these RNAi lines generate the glial wrapping
phenotype, these changes in glial number are unlikely to be
responsible for the failure of the PG to wrap the growing nerve.
The neural lamella is required by the perineurial
glia
Most integrins function through binding to the ECM, including
PS2PS integrin in Drosophila (Bokel and Brown, 2002).
Integrins are in turn important for ECM deposition, such as in
epithelial morphogenesis (Narasimha and Brown, 2004). However,
when PS integrin was knocked down in all glial layers using repoGAL4, Perlecan-GFP and -Laminin labeling appeared normal in
RNAi nerves as compared with control nerves (see Fig. S4 in the
supplementary material). This suggests that glial integrins are not
required for NL formation.
DEVELOPMENT
The knockdown of the integrin complex in glial cells caused
glial defects and decreased viability. For example, repo::PS-RNAi
animals died in late third instar and pupal stages. When UAS-CD8GFP was co-expressed to label the glial membrane, we observed
morphological changes paired with reduced intensity and changes
in the distribution of CD8-GFP (Fig. 5B). This suggests that PS
integrin is necessary in glial cells in the larval peripheral nerve.
However, repo-GAL4 is expressed in all three glial layers, which
are functionally and morphologically distinct. Therefore, to test for
integrin function in the individual glial layers, different GAL4
drivers were used to express integrin RNAi in specific glial
subtypes.
Integrins and glial development
RESEARCH ARTICLE 3819
Fig. 6. Overexpression of Mmp2 degrades the neural lamella and causes a perineurial glial wrapping defect. 46F-GAL4 was used to
express CD8-RFP (red) and UAS-Mmp2 in the PG. Expression was regulated by a temperature-sensitive GAL80 under the control of a ubiquitous
tubulin promoter (tubP-GAL80ts). The NL was labeled by Perlecan-GFP (green) and -Laminin (blue). The labeling and changes to the NL and PG are
also illustrated beneath. (A,B,F,G) Single 0.2m z-sections of the nerves; (C,H) projections of the entire z-stack. Boxed regions were digitally
expanded as shown in D and I. (E,J)Orthogonal sections of z-stacks from the positions indicated by the green lines. (A-E)In a control nerve,
Perlecan-GFP (green) and -Laminin (blue) labeled the outermost NL (arrows), which surrounds the CD8-RFP-labeled PG (red). Diffuse, low-level
Laminin staining was apparent within the nerve. (F-J)In a 46F::Mmp2 nerve, both Perlecan-GFP (green) and -Laminin immunolabeling (blue)
indicate that the remaining NL is partially detached from the surface of the nerve (arrows). -Laminin immunolabeling appeared to be increased
within the CD8-RFP-labeled (red) PG (arrowheads). The PG no longer wrapped around the entire circumference of the nerve and borders of distinct,
individual PG were identified. Scale bars: 10m in A-C,F-H; 5m in D,E,I,J.
(Fig. 6F and arrows in 6I,J), suggesting that overexpression of
Mmp2 in PG was sufficient to remove the NL. Surprisingly, high
levels of Laminin immunolabeling were observed within the PG
(Fig. 6I,J, arrowheads), suggesting that glia might upregulate
Laminin to compensate for the loss of the NL.
The PG (46F::CD8-RFP) expressing Mmp2 did not form a
complete circle around the nerve (Fig. 5H,J). Thus, disruption of
the ECM by Mmp2 generated a wrapping defect in the PG similar
to that of the PS knockdown in the 46F::PS-RNAi nerve.
Remarkably, the short-term expression of Mmp2 in the PG resulted
in a strong CNS phenotype in which the ventral nerve cord became
thin and elongated compared with that of controls (see Fig. S5 in
the supplementary material). This suggests a role of the ECM and
PG in maintaining nervous system morphology by constricting the
ventral nerve cord.
Integrin function is necessary for axon
ensheathment by wrapping glia
In the larval nerve, PS integrin is not only found in the outer glial
cells, but is also observed in the central regions where the WG
ensheath axons (Fig. 2E-G). To study integrin function in the WG,
the Nrv2-GAL4 driver was used to knock down PS in GFP-labeled
WG (Fig. 7). In wild-type nerves, the WG (n72) labeled with CD8GFP formed complex processes along the nerve (Fig. 7B; see Movie
3 in the supplementary material). The CD8-GFP labeling filled in the
DEVELOPMENT
The question is, then, whether integrin function in the PG relies
on the NL. The major components of the NL, such as Collagen IV,
Perlecan and laminins, are deposited by migrating hemocytes
during embryogenesis (Olofsson and Page, 2005; Stork et al.,
2008). Consistently, expression of perlecan (trol) and collagen IV
(viking) RNAi in glia did not affect or remove the NL (data not
shown). Instead, we chose to remove the entire ECM layer by
expressing a matrix metalloproteinase, Matrix metalloproteinase 2
(Mmp2), in glial cells. Mmp2 is one of two Mmp family members
in Drosophila (Page-McCaw et al., 2003) and is attached to the
extracellular membrane through a glycophosphatidylinositol (GPI)
anchor (Llano et al., 2002). Constitutive expression of Mmp2 by
repo-GAL4 or 46F-GAL4 caused lethality at embryonic and larval
first instar stages. Therefore, to study the function of the NL in later
stages, a temperature-sensitive inhibitor of GAL4 (GAL80ts) was
used. Embryos and larvae were raised at the permissive
temperature (18°C) and then, at the early third instar stage, Mmp2
expression was activated by transferring the larvae to the restrictive
temperature (29°C) for one day prior to dissection. The ECM was
labeled using Perlecan-GFP (or Collagen IV-GFP, data not shown)
and Laminin immunolabeling to determine the effectiveness of the
Mmp2 in disrupting the NL. In control nerves, Perlecan-GFP and
-Laminin were both found in the NL surrounding the PG (Fig. 6AE). When Mmp2 was expressed by the PG, Perlecan-GFP and
Laminin were greatly reduced and became detached from the PG
3820 RESEARCH ARTICLE
Development 138 (17)
Although PS had a clear function in the WG, we were unable
to detect the presence of ECM components in the internal regions
of the peripheral nerve. For example, Perlecan-GFP and VikingGFP (Collagen IV) were observed exclusively in the NL with no
internal labeling (Fig. 6A,F; data not shown). We also assayed the
distribution of Laminins. Drosophila has two alpha genes but only
one beta (LanB1) and one gamma (LanB2) gene. Strong beta and
gamma laminin expression was detected in the NL, but only
diffusely in internal regions of the nerve (Fig. 6B,D,E). However,
we cannot rule out the possibility that low levels of ECM might be
deposited internally given the diffuse laminin labeling we observed.
Therefore, to test whether an internal ECM is necessary for the
ensheathment of peripheral axons, Mmp2 was overexpressed in the
WG using Nrv2-GAL4. Unlike repo-GAL4 or 46F-GAL4,
constitutive expression of Mmp2 with Nrv2-GAL4 did not cause
lethality or a decrease in the WG processes in the peripheral nerve
(Fig. 7G,H). This suggests that the internal PS integrin might
function in the WG by binding to an unknown ligand that is not an
ECM component.
space between the HRP-labeled axons (Fig. 7A-C), as each WG
produces extensive processes that wrap around axons. In the
Nrv2::PS-RNAi larvae, CD8-GFP still formed processes in
between the HRP labeling and spread along the nerve. However, the
complexity of the processes was greatly decreased. For example, in
the PS-RNAi line (VDRC_GD29619), all WG (90/90) had severe
defects in which CD8-GFP labeling was observed in a single
longitudinal process (Fig. 7D-F; see Movie 4 in the supplementary
material). Importantly, this phenotype was repeated in the two PS
(mys) null MARCM clones that we were able to obtain in the WG
(data not shown). This suggests that the WG are unable to form or
maintain complex processes when PS integrin is reduced.
Integrin-ECM interactions are required for
perineurial glia function
PG form the outermost glial layer in the Drosophila nervous
system but their origin and function are not well understood, even
though they were identified some time ago (Edwards et al., 1993;
Schmid et al., 1999). Drosophila PG are structurally similar to their
vertebrate counterparts and have been proposed to have similar
roles (Lavery et al., 2007). Vertebrate perineurial cells and the
associated collagen fibers provide important mechanical support to
nerves and their development might rely on ECM-mediated
signals, as 1 integrin is found in the perineurium (Feltri et al.,
2002). However, little is known about the role of integrins in
perineurial cells.
Our results show that Drosophila PG express PS2 and PS
integrin subunits (and to a lesser extent PS3), which colocalize
with both Ilk and Talin. Knocking down integrins disrupts
perineurial wrapping, but we were unable to distinguish whether
the PG failed to initiate wrapping or failed to maintain their
DEVELOPMENT
Fig. 7. Expression of PS-RNAi in the wrapping glia. Nrv2-GAL4
was used to express CD8-GFP (green) and PS-RNAi in the WG. AntiHRP and DAPI labeling were used for axons (red) and nuclei (blue),
respectively. The labeling and phenotypes observed in the WG are
illustrated to the right. (A,D)Single 0.2m z-sections; (B,E,G,H)
projections of the entire z-stack; (C,F) orthogonal sections at the
positions indicated by the green lines. (A-C)In a control nerve, CD8-GFP
surrounded the DAPI-labeled glia nucleus (blue) and extended along
the nerve to fill complex processes (arrows) in between the HRP labeling
(red, arrowheads). (D-F)In an Nrv2::PS-RNAi nerve, CD8-GFP was
found only in a single process that extended along the labeled axons
(red, arrowheads). Arrows point to small projections and puncta of
CD8-GFP. (G,H)In an Nrv2::Mmp2 nerve, the CD8-GFP-labeled
processes of the WG appeared normal and spread throughout the
labeled axons, similar to the control nerve. Glial nuclei were identified
by Repo immunolabeling (blue in G). Scale bars: 10m in A,B,D,E,G,H;
5m in C,F.
DISCUSSION
The integrin complex in peripheral glia
Peripheral nerves in vertebrates and Drosophila are organized in
similar ways, with central axons wrapped by an inner class of glia
that are surrounded in turn by layers of external glia. The glia and
the surrounding ECM establish a tubular sheath to protect axons
from physical damage and pathogens (Parmantier et al., 1999). We
show that at least two integrin heterodimers are expressed in these
different glial layers and are localized at focal adhesions with Ilk
and Talin. We found that PS2PS integrin is prevalent in the PG
and that the PS3PS integrin is more prevalent in the WG. Since
PS2 integrin is expressed mostly in the outermost PG and can
bind ligands that contain the tripeptide RGD sequence (Bunch and
Brower, 1992; Fogerty et al., 1994), it most likely functions by
binding to ECM ligands in the NL. The majority of PS3 integrin
is expressed by the internal SPG and WG and PS3 integrin has
been shown to interact with laminins in Drosophila (Schock and
Perrimon, 2003; Stark et al., 1997). However, it is possible that the
integrin complex in the WG might have other ligands and might
mediate direct cell-cell interactions as we failed to detect a
pronounced basal lamina associated with the peripheral axons and
Mmp2 expression had no effect in the internal regions of the
peripheral nerves.
processes around the nerves to accommodate the growing nerve
surface. However, degradation of the NL by Mmp2 overexpression
generates a PG wrapping phenotype similar to that of PS RNAi.
Thus, binding of PS integrin to ligands in the ECM mediates the
radial spread of PG around the tubular structure of the nerve and
suggests that PG retract their membrane when integrin-ECM
interaction is interrupted. Moreover, knockdown of Talin in the PG
produces a similar wrapping defect. This suggests that, in the PG,
integrin adhesion complexes mediate the connection between the
extracellular NL and the intracellular actin cytoskeleton and are
required for the initiation or maintenance of glial ensheathment.
The function of the PG in the peripheral nerve is not well
understood. The PG do not generate an impermeable barrier and it
is the SPG layer that creates the blood-nerve barrier (Stork et al.,
2008). Loss of PG ensheathment did not result in paralysis or
lethality, which are signs of a disrupted blood-brain barrier
(Baumgartner et al., 1996; Schwabe et al., 2005). Larger molecules
(~500 kDa) are blocked by the NL or the PG (Stork et al., 2008),
perhaps mirroring the protective function of the vertebrate
perineurium against pathogens (Parmantier et al., 1999). However,
in Mmp2-overexpressing larvae, the affected nerves become thin
and are difficult to retain intact during tissue preparation. This
suggests that the NL and PG provide important mechanical support
as is seen with the perineurium in mammalian nerves (Parmantier
et al., 1999).
PS integrin is required to maintain wrapping glia
ensheathment of the peripheral axons
In the center of Drosophila peripheral nerves, the WG embed
axons in bundles or individually within single membrane wraps,
similar to the non-myelinating Schwann cells of vertebrate
peripheral nerves (Corfas et al., 2004). Our results show that WG
predominantly express PS3 and PS integrin subunits in
complexes positive for Ilk and Talin along the WG membrane.
When PS expression is knocked down, the complexity of the glial
processes between the associated axons is greatly reduced. Only a
few long processes and small membrane protrusions are observed
around the axons, suggesting that the WG might be retracting their
processes in the absence of PS integrin. The role of integrins
appears to be conserved between WG and Schwann cells in
vertebrates. For example, myelinating Schwann cells lacking 1
integrin or Ilk do not extend membrane processes around axons,
resulting in impaired radial sorting (Feltri et al., 2002; Pereira et
al., 2009). The role of integrins in non-myelinating Schwann cells
is not known but our results suggest that integrins have similar
functions in Drosophila and vertebrates in mediating the glial
ensheathment of peripheral axons.
No clear ECM has been observed in the internal regions of
Drosophila nerves by immunofluorescence analysis or transmission
electron microscopy (Lavery et al., 2007; Stork et al., 2008).
Therefore, the integrin complex could promote WG sheath
formation by mediating direct cell-cell adhesion between the glial
membrane and its associated axon, or between glial membranes. A
potential candidate for an integrin-interacting protein expressed on
axons or glia is Neuroglian, the Drosophila L1 (Nrcam) homolog.
L1 is an Ig domain transmembrane protein that is known to bind
RGD-dependent integrins (Ruppert et al., 1995; Montgomery et al.,
1996). Loss of the integrin-binding domain of L1 results in
wrapping defects in both myelinating and non-myelinating
Schwann cells (Itoh et al., 2005). However, we cannot rule out the
presence of low levels of ECM given the weak laminin
immunolabeling that we observed. Even though Mmp2 expression
RESEARCH ARTICLE 3821
in the WG had no effect, it is still possible that the integrinmediated adhesion is Mmp2 resistant. To resolve this issue, further
ultrastructural and genetic studies will be required.
Acknowledgements
We thank Dr Shigeo Hayashi for generously providing antibodies; Drs Guy
Tanentzapf, Christian Klämbt, Doug Allan, the Bloomington and DGRC Stock
Centers for fly stocks; the TRiP at Harvard Medical School (NIH/NIGMS R01GM084947) for providing transgenic RNAi fly stocks; Doug Allan, Patrick
Cafferty, Mary Gilbert and Guy Tanentzapf for helpful discussions and
comments on the manuscript. X.X. was supported by a scholarship from the
Multiple Sclerosis Society of Canada. This study was supported by grants to
V.J.A. from the Canadian Institutes of Health Research and the Natural Science
and Engineering Research Council of Canada.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.064816/-/DC1
References
Alfonso, T. B. and Jones, B. W. (2002). gcm2 promotes glial cell differentiation
and is required with glial cells missing for macrophage development in
Drosophila. Dev. Biol. 248, 369-383.
Baumgartner, S., Littleton, J. T., Broadie, K., Bhat, M. A., Harbecke, R.,
Lengyel, J. A., Chiquet-Ehrismann, R., Prokop, A. and Bellen, H. J. (1996).
A Drosophila neurexin is required for septate junction and blood-nerve barrier
formation and function. Cell 87, 1059-1068.
Bhat, M. (2003). Molecular organization of axo-glial junctions. Curr. Opin.
Neurobiol. 13, 552-559
Bokel, C. and Brown, N. H. (2002). Integrins in development: moving on,
responding to, and sticking to the extracellular matrix. Dev. Cell 3, 311-321.
Brower, D. L., Wilcox, M., Piovant, M., Smith, R. J. and Reger, L. A. (1984).
Related cell-surface antigens expressed with positional specificity in Drosophila
imaginal discs. Proc. Natl. Acad. Sci. USA 81, 7485-7489.
Brown, N. H., Gregory, S. L. and Martin-Bermudo, M. D. (2000). Integrins as
mediators of morphogenesis in Drosophila. Dev. Biol. 223, 1-16.
Bunch, T. A. and Brower, D. L. (1992). Drosophila PS2 integrin mediates RGDdependent cell-matrix interactions. Development 116, 239-247.
Bunch, T. A., Salatino, R., Engelsgjerd, M. C., Mukai, L., West, R. F. and
Brower, D. L. (1992). Characterization of mutant alleles of myospheroid, the
gene encoding the beta subunit of the Drosophila PS integrins. Genetics 132,
519-528.
Chen, Z. L. and Strickland, S. (2003). Laminin gamma1 is critical for Schwann cell
differentiation, axon myelination, and regeneration in the peripheral nerve. J.
Cell Biol. 163, 889-899.
Corfas, G., Velardez, M. O., Ko, C. P., Ratner, N. and Peles, E. (2004).
Mechanisms and roles of axon-Schwann cell interactions. J. Neurosci. 24, 92509260.
Delon, I. and Brown, N. H. (2007). Integrins and the actin cytoskeleton. Curr.
Opin. Cell Biol. 19, 43-50.
Dietzl, G., Chen, D., Schnorrer, F., Su, K. C., Barinova, Y., Fellner, M., Gasser,
B., Kinsey, K., Oppel, S., Scheiblauer, S. et al. (2007). A genome-wide
transgenic RNAi library for conditional gene inactivation in Drosophila. Nature
448, 151-156.
Edwards, J. S., Swales, L. S. and Bate, M. (1993). The differentiation between
neuroglia and connective tissue sheath in insect ganglia revisited: the neural
lamella and perineurial sheath cells are absent in a mesodermless mutant of
Drosophila. J. Comp. Neurol. 333, 301-308.
Feltri, M. L. and Wrabetz, L. (2005). Laminins and their receptors in Schwann
cells and hereditary neuropathies. J. Peripher. Nerv. Syst. 10, 128-143.
Feltri, M. L., Graus Porta, D., Previtali, S. C., Nodari, A., Migliavacca, B.,
Cassetti, A., Littlewood-Evans, A., Reichardt, L. F., Messing, A., Quattrini,
A. et al. (2002). Conditional disruption of beta 1 integrin in Schwann cells
impedes interactions with axons. J. Cell Biol. 156, 199-209.
Fernandez-Valle, C., Gwynn, L., Wood, P. M., Carbonetto, S. and Bunge, M.
B. (1994). Anti-beta 1 integrin antibody inhibits Schwann cell myelination. J.
Neurobiol. 25, 1207-1226.
Fogerty, F. J., Fessler, L. I., Bunch, T. A., Yaron, Y., Parker, C. G., Nelson, R. E.,
Brower, D. L., Gullberg, D. and Fessler, J. H. (1994). Tiggrin, a novel
Drosophila extracellular matrix protein that functions as a ligand for Drosophila
alpha PS2 beta PS integrins. Development 120, 1747-1758.
Freeman, M. R. and Doherty, J. (2006). Glial cell biology in Drosophila and
vertebrates. Trends Neurosci. 29, 82-90.
DEVELOPMENT
Integrins and glial development
Itoh, K., Fushiki, S., Kamiguchi, H., Arnold, B., Altevogt, P. and Lemmon, V.
(2005). Disrupted Schwann cell-axon interactions in peripheral nerves of mice
with altered L1-integrin interactions. Mol. Cell. Neurosci. 30, 624-629.
Karpova, N., Bobinnec, Y., Fouix, S., Huitorel, P. and Debec, A. (2006). Jupiter,
a new Drosophila protein associated with microtubules. Cell Motil. Cytoskel. 63,
301-312.
Kelso, R. J., Buszczak, M., Quinones, A. T., Castiblanco, C., Mazzalupo, S.
and Cooley, L. (2004). Flytrap, a database documenting a GFP protein-trap
insertion screen in Drosophila melanogaster. Nucleic Acids Res. 32, D418-D420.
Lavery, W., Hall, V., Yager, J. C., Rottgers, A., Wells, M. C. and Stern, M.
(2007). Phosphatidylinositol 3-kinase and Akt nonautonomously promote
perineurial glial growth in Drosophila peripheral nerves. J. Neurosci. 27, 279288.
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible cell marker for
studies of gene function in neuronal morphogenesis. Neuron 22, 451-461.
Llano, E., Adam, G., Pendas, A. M., Quesada, V., Sanchez, L. M., Santamaria,
I., Noselli, S. and Lopez-Otin, C. (2002). Structural and enzymatic
characterization of Drosophila Dm2-MMP, a membrane-bound matrix
metalloproteinase with tissue-specific expression. J. Biol. Chem. 277, 2332123329.
Martin-Bermudo, M. D., Alvarez-Garcia, I. and Brown, N. H. (1999). Migration
of the Drosophila primordial midgut cells requires coordination of diverse PS
integrin functions. Development 126, 5161-5169.
McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K. and Davis, R. L. (2003).
Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, 17651768.
Montgomery, A. M., Becker, J. C., Siu, C. H., Lemmon, V. P., Cheresh, D. A.,
Pancook, J. D., Zhao, X. and Reisfeld, R. A. (1996). Human neural cell
adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v
beta 3. J. Cell Biol. 132, 475-485.
Morin, X., Daneman, R., Zavortink, M. and Chia, W. (2001). A protein trap
strategy to detect GFP-tagged proteins expressed from their endogenous loci in
Drosophila. Proc. Natl. Acad. Sci. USA 98, 15050-15055.
Narasimha, M. and Brown, N. H. (2004). Novel functions for integrins in
epithelial morphogenesis. Curr. Biol. 14, 381-385.
Newbern, J. and Birchmeier, C. (2010). Nrg/ErbB sigaling networks in Schwann
cell development and myelination. Semin. Cell Dev. Biol. 21, 922-928.
Olofsson, B. and Page, D. T. (2005). Condensation of the central nervous system
in embryonic Drosophila is inhibited by blocking hemocyte migration or neural
activity. Dev. Biol. 279, 233-243.
Olsson, Y. (1990). Microenvironment of the peripheral nervous system under
normal and pathological conditions. Crit. Rev. Neurobiol. 5, 265-311.
Page-McCaw, A., Serano, J., Sante, J. M. and Rubin, G. M. (2003). Drosophila
matrix metalloproteinases are required for tissue remodeling, but not embryonic
development. Dev. Cell 4, 95-106.
Parker, R. J. and Auld, V. J. (2006). Roles of glia in the Drosophila nervous
system. Semin. Cell Dev. Biol. 17, 66-77.
Parmantier, E., Lynn, B., Lawson, D., Turmaine, M., Namini, S. S.,
Chakrabarti, L., McMahon, A. P., Jessen, K. R. and Mirsky, R. (1999).
Schwann cell-derived Desert hedgehog controls the development of peripheral
nerve sheaths. Neuron 23, 713-724.
Pereira, J. A., Benninger, Y., Baumann, R., Goncalves, A. F., Ozcelik, M.,
Thurnherr, T., Tricaud, N., Meijer, D., Fassler, R., Suter, U. et al. (2009).
Integrin-linked kinase is required for radial sorting of axons and Schwann cell
remyelination in the peripheral nervous system. J. Cell Biol. 185, 147-161.
Development 138 (17)
Perkins, A. D., Ellis, S. J., Asghari, P., Shamsian, A., Moore, E. D. and
Tanentzapf, G. (2010). Integrin-mediated adhesion maintains sarcomeric
integrity. Dev. Biol. 338, 15-27.
Ruppert, M., Aigner, S., Hubbe, M., Yagita, H. and Altevogt, P. (1995). The L1
adhesion molecule is a cellular ligand for VLA-5. J. Cell Biol. 131, 1881-1891.
Schmid, A., Chiba, A. and Doe, C. Q. (1999). Clonal analysis of Drosophila
embryonic neuroblasts: neural cell types, axon projections and muscle targets.
Development 126, 4653-4689.
Schock, F. and Perrimon, N. (2003). Retraction of the Drosophila germ band
requires cell-matrix interaction. Genes Dev. 17, 597-602.
Schwabe, T., Bainton, R. J., Fetter, R. D., Heberlein, U. and Gaul, U. (2005).
GPCR signaling is required for blood-brain barrier formation in Drosophila. Cell
123, 133-144.
Sepp, K. J. and Auld, V. J. (1999). Conversion of lacZ enhancer trap lines to GAL4
lines using targeted transposition in Drosophila melanogaster. Genetics 151,
1093-1101.
Sepp, K. J., Schulte, J. and Auld, V. J. (2000). Developmental dynamics of
peripheral glia in Drosophila melanogaster. Glia 30, 122-133.
Sepp, K. J., Schulte, J. and Auld, V. J. (2001). Peripheral glia direct axon
guidance across the CNS/PNS transition zone. Dev. Biol. 238, 47-63.
Stark, K. A., Yee, G. H., Roote, C. E., Williams, E. L., Zusman, S. and Hynes,
R. O. (1997). A novel alpha integrin subunit associates with betaPS and
functions in tissue morphogenesis and movement during Drosophila
development. Development 124, 4583-4594.
Stork, T., Engelen, D., Krudewig, A., Silies, M., Bainton, R. J. and Klambt, C.
(2008). Organization and function of the blood-brain barrier in Drosophila. J.
Neurosci. 28, 587-597.
Sun, B., Xu, P. and Salvaterra, P. M. (1999). Dynamic visualization of nervous
system in live Drosophila. Proc. Natl. Acad. Sci. USA 96, 10438-10443.
Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A. and Driscoll, M.
(2000). Heritable and inducible genetic interference by double-stranded RNA
encoded by transgenes. Nat. Genet. 24, 180-183.
Wada, A., Kato, K., Uwo, M. F., Yonemura, S. and Hayashi, S. (2007).
Specialized extraembryonic cells connect embryonic and extraembryonic
epidermis in response to Dpp during dorsal closure in Drosophila. Dev. Biol. 301,
340-349.
Wallquist, W., Plantman, S., Thams, S., Thyboll, J., Kortesmaa, J.,
Lannergren, J., Domogatskaya, A., Ogren, S. O., Risling, M.,
Hammarberg, H. et al. (2005). Impeded interaction between Schwann cells
and axons in the absence of laminin alpha4. J. Neurosci. 25, 3692-3700.
Xiong, W. C., Okano, H., Patel, N. H., Blendy, J. A. and Montell, C. (1994).
repo encodes a glial-specific homeo domain protein required in the Drosophila
nervous system. Genes Dev. 8, 981-994.
Yang, D., Bierman, J., Tarumi, Y. S., Zhong, Y. P., Rangwala, R., Proctor, T. M.,
Miyagoe-Suzuki, Y., Takeda, S., Miner, J. H., Sherman, L. S. et al. (2005).
Coordinate control of axon defasciculation and myelination by laminin-2 and -8.
J. Cell Biol. 168, 655-666.
Yu, W. M., Feltri, M. L., Wrabetz, L., Strickland, S. and Chen, Z. L. (2005).
Schwann cell-specific ablation of laminin gamma1 causes apoptosis and
prevents proliferation. J. Neurosci. 25, 4463-4472.
Yu, W. M., Yu, H., Chen, Z. L. and Strickland, S. (2009). Disruption of laminin in
the peripheral nervous system impedes nonmyelinating Schwann cell
development and impairs nociceptive sensory function. Glia 57, 850-859.
DEVELOPMENT
3822 RESEARCH ARTICLE